Vector borne flaviviruses, which belong to the Family Flaviviridae, genus Flavivirus, comprise some of the most important emerging and re-emerging viral pathogens. The tick borne flaviviruses (TBFV) include tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Alkhurma hemorrhagic fever virus, Powassan/deer tick virus (POWV/DTV) and Langat virus (LGTV). TBFV are generally transmitted to humans by ixodid ticks, and cause a spectrum of disease ranging from mild febrile illness to encephalitis, meningitis or hemorrhagic fevers. In the US, the cases of all tick-borne infections are increasing dramatically. POWV/DTV are the only autochthonous TBFV and case reports of infection are also on the increase. The mosquito borne flaviviruses include West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). The dramatic and ongoing pandemic attributed to the MBFV Zika virus is important because of its capacity to cause a severe congenital Zika disease as well an ever increasing spectrum of neurological syndromes in adults. Our current research is focused on the TBFV, but studying the biology of TBFV will elucidate the biology of other vector borne viruses. The research in our laboratory employs virology, immunology, entomology, advanced imaging techniques, genomics, cell biology, molecular biology, and vector biology. We primarily study LGTV, a naturally attenuated member of the TBFV that can be safely studied at Biosafety Level-2 (BSL-2) making it an excellent model to gain insight into the TBFV. In addition, we also study the virulent autochthonous BSL-3 POWV/DTV. Neither of these 2 agents are Select Agents which greatly facilitate research studies. In addition, we have also begun to study the BSL2 MBFV, Zika virus. With the recent emergence of Zika in Latin America and the US, similar avenues of inquiry are being explored for that vexatious pathogen. Comparison of VBFV cytoarchitecture in mammalian and arthropod cells. Competing priorities and limited personnel have made it difficult to pursue this long term aspect of the BVBV research agenda in the current cycle. TBFV infection in ex vivo cultures of I. scapularis organs. Infection in ticks is patently a critical feature of TBFV biology, but it is woefully understudied. We developed an ex vivo system in which salivary glands and midgut are dissected out and can be successfully cultured for about a week. We showed that replication and spread of TBFV occurs in these ex vivo cultures, and we are presently at work identifying the specific acini and cell types in the SG cultures that are infected. These ex vivo tick organ cultures represent a gateway technology that is proving amenable not only to the study of viral infection but also to determining the role of various tick genes in virus infection in this critical tick organ. Molecular biology and molecular pathogenesis of acute and persistent VBFV infection. The role of persistent infection in natural life cycle of TBFV in rodent and arthropod hosts is not well characterized, but may be responsible for prolonged debilitating sequelae observed in survivors of acute TBFV infection. Experimental work by others on MBFV and the recent findings that ZIKV can persist in human semen and other sites for extended periods of time strongly imply that persistence of both TBFV and MBFV may be an underappreciated feature of those infections. LGTV infection causes an acute lytic crisis of most cells, and we have shown the mechanism of cell death is apoptosis. The surviving cells somehow evade apoptosis and go on to establish persistent infection. A signature of 451 genes was associated with the initiation of persistence. Intensive bioinformatics perusal using Ingenuity Pathway Analysis and other similar software packages, revealed that networks associated with cell survival were playing a role. The acute phase was associated with networks and pathways. Work on defining the role of several pro-survival genes is nearing completion. The unfolded protein response or UPR is another cellular mechanism that is designed to protect the cell from excess production of proteins. Since flavivirus infection is associated with the synthesis of a massive amount of viral proteins, these viruses trigger the UPR. In order to replicate, the viruses must devise strategies to circumvent the UPR. We are focusing on the PERK arm of the UPR. We previously published that the I. scapularis derived ISE-6 cell line develops a persistent TBFV infection with no apparent acute phase and with no evidence of DI particles being involved. A transcriptomic analysis of infected ISE6 cells is being prepared for publication. TBFV infection in the reservoir host. Peromyscus leucopus is the identified reservoir host for POWV. Parallel POWV infection of Pl and Balb/C and C57Bl/6 mice yielded surprising results which we published this year. POWV causes a fulminant and lethal neurological infection in both strains of lab mice after either intracerebral or intraperitoneal infection. However, the Peromyscus mice show no overt signs of disease after either ic or ip inoculation. Detailed studies revealed that virus replication is severely restricted in time and place to the olfactory bulb and projecting tracts. Only a short-lived and very low level viremia occurs but a neutralizing antibody response does develop. A detailed in vivo analysis of the cellular transcriptome revealed that the PL mice mount a robust antiviral response through the interferon system, but that alterations in cholesterol metabolism are also evident. The role of novel non-interferon genes also seems likely. The fact that a known reservoir species fails to succumb or even become viremic, raises interesting questions concerning tick-to-tick transmission.